UNIVERSITÀ DEGLI STUDI DI MILANO Scuola di Dottorato in Scienze Biochimiche, Nutrizionali e Metaboliche Dipartimento di Scienze Farmacologiche e Biomolecolari Dottorato di Ricerca in Biochimica XXVI ciclo BIO/10 Cholesterol Homeostasis: involvement of histone deacetylases in the molecular regulatory pathway of Cholesterol 7α-hydroxylase Erika FIORINO Matr. R09261 Tutor: Prof. Maurizio CRESTANI Coordinatore: Prof. Francesco BONOMI Anno accademico 2013-2014
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UNIVERSITÀ DEGLI STUDI DI MILANO
Scuola di Dottorato in Scienze Biochimiche, Nutrizionali e Metaboliche
Dipartimento di Scienze Farmacologiche e Biomolecolari
Q-PCR determination of Cyp7a1 mRNA expression on murine primary hepatocytes.
Cells were transducted with the respective Ad (100 MOI) and compared to control
AdLacz. RNA was extracted 72h after transduction. Cyp7a1 mRNA levels are 7 fold
increased by AdHdac1 compared to the empty vector AdLacZ, 8 fold by AdSmrt
and 21 fold by AdHdac7 alone or in co-treatment with AdHdac 4, and 5. Data are
expressed as mean ± SD and are normalized on 36b4 as reference gene. ANOVA
statistical analysis was performed with Dunnett’s Multiple Comparison post test vs
AdLacZ; * indicates statistical significance with p<0.05; ** indicates statistical
significance with p<0.01.
AdHdac4,5,7
AdLacZ AdHdac1 AdHdac4 AdHdac5 AdHdac7 AdSmrt0
2
5
10
15
20
25
30
35
**
**
**
*** *R
ela
tive
exp
ress
ion
(arb
itra
ry u
nit
s)CYP7A1
Results
67
Hdac7 and Smrt SILENCING SIGNIFICANTLY INCREASES
Cyp7a1 EXPRESSION in vivo
To investigate the acute effect of silencing Hdacs and Smrt, I amplified
and purified the Ad used in the experiment described above to proceed with the
in vivo treatment. C57BL/6J mice were divided in 4 groups, one for each
treatment, and injected in the jugular vein with 6*109 pfu of AdLacz, AdHdac1,
AdHdac7 and AdSmrt.
Body weight of mice at the beginning (B.I.) and at the end (A.I.) of the Ad
infection (at sacrifice) did not yield any difference neither among the groups of
treatment nor between B.I. and A.I. (Fig. 21), meaning that Ad did not affect
health of infected mice.
Fig. 21: Mice body weight
Mice body weight before (B.I.) and after (A.I.) infection with Ad in C57BL/6J mice.
Data are expressed as mean ± SD. TWO WAY ANOVA statistical analysis was
performed and no statistically significant difference was observed.
Real Time PCR analysis of total RNA from liver extracts of mice infected
with Ad revealed that AdHdac1 (Fig. 22A) was ineffective in silencing its target
gene, whose expression did not significantly differ from control AdLacz. AdHdac7
and AdSmrt infections, instead, efficiently reduced Hdac7 and Smrt expression
B.I. A.I. B.I. A.I. B.I. A.I. B.I. A.I.0
10
20
30
AdLacz AdHdac1 AdHdac7 AdSmrt
Body w
eig
ht
(g)
Results
68
respectively (Fig. 22B, C). Therefore, I measured the contribution of these
knock-down on Cyp7a1 expression and I found that AdHdac1, consistent with its
inability in silencing its target gene, did not affect the expression of the gene,
while AdHdac7 and AdSmrt significantly increased Cyp7a1 mRNA levels (Fig. 23),
confirming their important role in the regulation of BA metabolism also in vivo.
Fig. 22: Hdac1, Hdac7 and Smrt silencing in C57BL/6J mice
Relative expression of genes coding Hdac1, Hdac7 and Smrt measured by Real-
Time PCR. Mice livers collected 5 days after intra-jugular injection with the
respective Ad (6*109 pfu) were compared to to control AdLacz. Data are expressed
as mean ± SD and are normalized on 36b4 as reference gene. Student’s t test
statistical analysis was performed; * indicates statistical significance with p<0.05;
** indicates statistical significance with p<0.01.
Fig. 23: Cyp7a1 gene expression levels in C57BL/6J mice treated with
AdHDACs and AdSmrt
Q-PCR determination of Cyp7a1 mRNA expression on liver extracts of mice
infected with AdHDACs and AdSmrt. Mice livers collected 5 days after intra-jugular
injection with the respective Ad (6*109 pfu) were compared to control AdLacz.
Data are expressed as mean ± SD and are normalized on 36b4 as reference gene.
Student’s t test statistical analysis was performed; * indicates statistical
significance with p<0.05; *** indicates statistical significance with p<0.001.
HDAC1
AdLacz AdHdac10
0.5
1.0
1.5
mR
NA levels
(arb
itra
ry u
nit
s)
A HDAC7
AdLacz AdHdac70
0.5
1.0
1.5
*
mR
NA levels
(arb
itra
ry u
nit
s)
B SMRT
AdSMRT0
0.5
1.0
1.5
**
mR
NA
levels
(arb
itra
ry u
nit
s)
AdLacz
C
CYP7A1
AdLacz AdHdac1
0
0.5
1.0
1.5
2.0
2.5
Rela
tive e
xpre
ssio
n(a
rbit
rary
unit
s)
CYP7A1
AdLacz AdSmrt
0
0.5
1.0
1.5
2.0
2.5***
Rela
tive e
xpre
ssio
n(a
rbit
rary
unit
s)
CYP7A1
AdLacz AdHdac7
0
0.5
1.0
1.5
*
Rela
tive e
xpre
ssio
n(a
rbit
rary
unit
s)
A B C
Results
69
Next, I measured total plasma cholesterol in these mice and, as expected,
I did not observe differences among the groups, most likely because a 5 days
treatment was not sufficient to elicit significant changes in cholesterol
metabolism (Fig. 24).
Fig. 24: Plasma
Cholesterol levels
after Ad treatments
Plasma Cholesterol
levels after AdHdacs
and AdSmrt treatment
at sacrifice reveale any
significant change
among the groups. Data
are expressed as mean
± SD. ANOVA statistical
analysis was
performed.
HDAC7 DELETION INDUCE PHENOTYPICAL IMPROVEMENT
ON CHOLESTEROL and LIPOPROTEIN PROFILE of H7LivKO
mice
Previous investigations performed in our laboratory demonstrated that
HDAC7 shuttles from the cytoplasm to the nucleus in hepatic cells treated with
BA, and subsequently is recruited to CYP7A1 promoter as part of the repressive
complex. This suggests a central role of HDAC7 in the inhibitory feedback of
CYP7A1 mediated by BA and consequently on cholesterol homeostasis (Mitro et
al., 2007). Therefore, since it was shown that the total body knock-out is not
viable, to investigate the role of HDAC7 in vivo I generated HDAC7 liver-specific
knock-out mice (H7LivKO) with the Cre/Lox technology. Mice were challenged
Plasma Cholesterol
AdLacz AdHdac1 AdHdac7 AdSmrt0
20
40
60
80
100
mg
/dL
Results
70
with Western Diet, a diet enriched in cholesterol with the aim to emphasize the
possible effect of Hdac7 deletion on cholesterol and BA metabolism.
Actual deletion of Hdac7 was assessed by real time qPCR of liver genomic
DNA vs non hepatic genomic DNA. H7LivKO mice with excision percentage lower
than 60% were excluded from the study (Fig. 25). Moreover, Hdac7 deletion was
confirmed by immunofluorescence analysis observed with confocal microscopy
(Fig. 26).
Fig. 25: Hdac7 percentage of excision
Excision efficiency by Cre recombinase on LoxP sites
insered into Hdac7 gene in the liver was evaluated
comparing liver expression to tail expression, in
which the gene is not deleted.
Fig. 26: Hdac7 deletion in H7LivKO
Immunofluorescence analysis of Hdac7 on liver slices of H7LivKO and wild type
mice on western diet.
CTRL
H7LivKO
HDAC7 NUCLEI MERGE
%EXCISION
CTRL KO-50
0
50
100
Results
71
REDUCED BODY WEIGHT in H7LivKO on Western Diet
Body weight analysis showed 12% statistically significant reduction in
H7LivKO mice compared to wild type mice (Fig. 27A), despite the equal food
consumption (Fig. 27B), suggesting a possible role of HDAC7 on lipid metabolism.
Fig. 27: Body weight reduction in H7LivKO mice fed WD:
Body weight (A) and food consumption (B) in H7LivKO mice compared to wild type
mice after 16 weeks on Western diet. Data are expressed as mean ± SD. Student’s
t test statistical analysis was performed; * indicates statistical significance with
p<0.05.
REDUCED LDL-CHOLESTEROL in H7LivKO on Western Diet
Mice on Western diet (WD) showed total plasma cholesterol levels
significantly higher than mice on Chow diet (CD); moreover, I observed a
statistically significant decrease of total plasma cholesterol, equal to 11%, in
H7LivKO mice compared to wild type (wt) mice (Fig 28A).
In light of this evidence, I decided to better characterize this difference
by performing Fast Protein Liquid Chromatography (FPLC) to analyze cholesterol
levels in the different lipoprotein fractions. The FPLC analysis revealead a
reduction of LDL-Cholesterol in H7LivKO mice compared to the respective
controls (Fig. 28B).
Food consumption
CTRL H7LivKO0
1
2
3
(g/m
ou
se/d
ay)
CTRL H7LivKO0
10
20
30
40
*
Body weight
(g)
-12%
A B
Results
72
Fig. 28: Plasma cholesterol analysis on H7LivKO mice fed WD
A) Total plasma cholesterol levels of control mice on WD are significantly higher
than mice on CD. 11% decrease of total plasma cholesterol is observed in mice
lacking HDAC7 in the liver compared to control on WD. Data are expressed as
mean ± SD. Student’s t test statistical analysis was performed; * indicates
statistical significance with p<0.05.
B) Fast Protein Liquid Chromatography revealed a reduced amount in LDL-
cholesterol in H7LivKO mice.
REDUCED LIVER LIPID ACCUMULATION and LIVER CHOLESTEROL in H7LivKO on Western Diet
We next analyzed the hepatic lipid content in wt and H7LivKI mice.
Histological analysis by hematoxylin and eosin staining on liver slices highlighted
lower lipid accumulation in H7LivKO compared to wt mice (Fig. 29).
Fig. 29: Liver lipid
accumulation
8 µm liver sections
with hematoxylin
and eosin staining
of H7LivKO (right)
and wyld type mice
(left) on WD.
Cholesterol
10 20 30 40 500
50
100
150
VLDL
LDL
HDL
FRACTIONS
mg/d
l
CTRL
H7LivKO
Plasma Cholesterol
CTRL H7LivKO0
100
200
300-11%
*
mg/d
l
CTRL
ChowDiet
***
WesternDiet
A B
CTRL H7LivKO
Results
73
I extracted and quantified triglyceride and cholesterol levels, typical
components of lipid droplets observed in hepatic steatosis. Surprisingly, liver
triglycerides were reduced in H7LivKO though the difference did not reach
statistical significance (Fig. 30A). On the other hand, I observed a statistically
significant reduction of hepatic cholesterol levels in H7LivKO mice compared to
controls (Fig. 30B).
Fig. 30: Hepatic lipid analysis
A) Liver Triglycerides quantification after lipid extraction by methanol:chloroform
using 3H-triolein as internal standard. Data are expressed as mean ± SD.
B) Liver Cholesterol quantification after lipid extraction by methanol:chloroform
using 3H-triolein as internal standard. Data are expressed as mean ± SD. Student’s
t test statistical analysis was performed; * indicates statistical significance with
p<0.05.
INCREASED Liver BILE ACIDS and Cyp7A1 in H7LivKO on Western Diet
Consistent with hepatic cholesterol decrease in H7LivKO mice, I also
observed increased liver BA in knock out vs wt mice. Consistent with these
results, Cyp7a1 expression was higher in H7LivKO vs wt mice (Fig. 31A e B),
suggesting that upregulation of the rate-limiting enzyme in BA synthesis resulted
in increased BA production.
CTRL H7LivKO0
100
200
300
Liver
Triglycerides
ug t
rigl/
mg liv
er
A
CTRL H7LivKO0
10
20
30
40
50
Liver
Cholesterol
*ug c
hol/
mg liv
er
B
Results
74
Fig. 31: Increased Liver BILE ACIDS and Cyp7A1 in H7LivKO on Western Diet
A) Liver BA quantification after lipid extraction by methanol:chloroform using 3H-
triolein as internal standard. Data are expressed as mean ± SD. Student’s t test
statistical analysis was performed; * indicates statistical significance with p<0.05.
B) Q-PCR determination of Cyp7a1 mRNA expression of liver extracts. Data are
expressed as mean ± SD and are normalized on 36b4 as housekeeping gene.
Student’s t test statistical analysis was performed; * indicates statistical
significance with p<0.05.
INCREASED LIVER, PLASMA and FECAL BA in H7LivKO on Western Diet
To better define the impact of Hdac7 ablation on BA metabolism, I
performed a more detailed analysis of BA by LC-MS/MS for the quantitation of
endogenous BA in plasma, liver and feces. Unconjugated BA were significantly
increased in plasma of H7LivKO mice compared to wt mice (Fig. 32 A). The
analysis of hepatic BA showed increased muricholic acids (MCA), α-MCA and β-
MCA and ursodeoxycholic acid (UDCA), three hydrophilic BA that are antagonists
of the BA nuclear receptor FXR (Fig. 32 B). With the exception of cholic acid
(CA), lithocholic acid (LCA) and UDCA, the content of the most Almost all the
most abundant fecal BA, an index of increased BA biosynthesis, was higher in
H7LivKO mice (Fig. 32 C).
CTRL H7LivKO0
0.5
1.0
1.5 Cyp7A1*
Re
lati
ve e
xpre
ssio
n(a
rbit
rary
un
its)
CTRL H7LivKO0
20
40
60
80 Liver BA
*
nm
olB
A/m
g liv
er
A B
Results
75
Fig. 32: LC-MS/MS based assay on plasma, liver and feces
BA quantitation of plasma and liver and feces extracts. Data are expressed as
mean ± SD. Student’s t test statistical analysis was performed; * indicates
statistical significance with p<0.05; ** indicates statistical significance with
p<0.01.
Plasma
0.0
0.5
1.0
1.5
Wild type
HDAC7LIVKO
P=0.07
*
*
****
** ******
P=0.051
P=0.07Undete
rmin
ed
Undete
rmin
ed
Undete
rmin
ed
mM
A
Liver
0.0
0.5
1.0
3
13
23Wild type
HDAC7LIVKO
Undete
rmin
ed
Undete
rmin
ed
Undete
rmin
ed
Undete
rmin
ed
*
*
*** ********
***
***ng/m
g o
fti
ssue
B
Feces
0.0
0.5
1.0
1.5Wild type
HDAC7LIVKO
Undete
rmin
ed
Undete
rmin
ed
Undete
rmin
ed
Undete
rmin
ed
Undete
rmin
ed
Undete
rmin
ed
Undete
rmin
ed
***
******
mo
li/g
of
Sto
ols
C
Results
76
H7LivKO SHOW DIFFERENT HDL-CHOLESTEROL PROFILE, RICHER in SMALLER HDL
In depth analysis of lipoprotein subfractions brought up to our attention a
different population of HDLs. Since FPLC is an exclusion chromatography
technique, the size of molecules eluting in the late fractions, such as the ones
displayed in the blue circle of Fig. 33, should be smaller. Therefore, I
hypothesized that it might be a smaller population of HDL that is present only in
H7LivKO mice.
To better investigate this aspect, agarose electroforesis analysis was
performed. As shown in Fig. 34, plasma samples from control mice were more
electropositive and enriched in alpha HDL, containing more cholesterol. On the
other hand, plasma samples from H7LivKO mice migrated farther than controls,
suggesting that they contained smaller HDLs, probably resembling lipid poor pre-
beta HDLs.
Fig. 33: Fast Protein Liquid Chromatography revealed a smaller population of
HDL-cholesterol in H7LivKO mice
Cholesterol
10 20 30 40 500
50
100
150
VLDL
LDL
HDL
FRACTIONS
mg/
dl
CTRL
H7LivKO
Results
77
Fig. 34: Agarose electroforesis analysis of plasma lipoproteins
Ab anti ApoA-I was used for the detection of HDLs.
H7LivKOCTRL
DISCUSSION
Discussion
79
This study elucidates some peculiar aspects of the epigenetic
transcriptional regulation of CYP7A1, the rate-limiting enzyme for cholesterol
catabolism, deepening the important involvement of HDACs and corepressors in
this pathway. Several epidemiological studies have well established the
correlation between hypercholesterolemia and atherosclerosis and
cardiovascular disease risk. Great attention is turned to the discovery of new
pharmacological approaches aimed at reducing hypercholesterolemia to
overcome side effects of the drugs nowadays available and the poor response
that some patients develop towards these drugs. In this perspective, a deeper
understanding of the genetic and molecular mechanisms involved in the
pathophysiological regulation of cholesterol homeostasis is fundamental.
Quantitatively the most important route of cholesterol disposal in
mammals is BA biosynthesis (Chiang, 2002). This biosynthetic pathway represents
the final step of reverse cholesterol transport; in fact, excess of cholesterol
transported to the liver is catabolized to BA, which play an essential role in
maintaining cholesterol homeostasis. CYP7A1 is an enzyme expressed only in the
liver that represents the major check-point of BA biosynthesis and its impact on
cholesterol homeostasis has been fully demonstrated. The homozygous deletion
mutation in CYP7A1 locus in human patients, leading to the loss of the active site
and enzyme function, causes hypercholesterolemia with high levels of LDL
cholesterol (Pullinger et al., 2002). Moreover, Cyp7a1-tg mice show increased
hepatic cholesterol catabolism and increased BA pool size, improving high-fat
diet (HFD)–induced obesity, fatty liver and insulin resistance (Li et al., 2010a). In
light of this critical role in the maintenance of cholesterol homeostasis, in the
last decades many studies have been conducted to elucidate its regulatory
mechanisms. Expression of CYP7A1 gene is feedback inhibited by BA that act on
their nuclear receptor FXR that in the liver induces the expression of SHP
(Goodwin et al., 2000, Lu et al., 2000c) and in the intestine the release of FGF
15/19 (Inagaki et al., 2005), both leading to CYP7A1 inhibition.
As previously published by our laboratory, BA also repress CYP7A1 gene
transcription via an FXR-independent pathway, chronologically preceding the
Discussion
80
FXR-dependent mechanism. BA induce the dissociation of the coactivators PGC-
1α and CBP from HNF-4α, a master regulator of CYP7A1 (De Fabiani et al., 2003)
and simultaneously induce the recruitment of transcriptional corepressors such
as NcoR and SMRT, which create a repressive complex on CYP7A1 promoter
together with HDAC1, 3 and 7 (Mitro et al., 2007). These data highlighted
epigenetic regulation of cholesterol homeostasis. Epigenetic modifications of
chromatin induced by HDACs activity modulate gene transcription, impairing
transcriptional activators accessibility to DNA (López-Rodas et al., 1993b,
Grunstein, 1997a) and are associated to several pathological conditions, such as
cancer (Miremadi et al., 2007), insulin resistance and obesity (Xiang et al., 2004,
Gray and Ekström, 2001). In recent years a growing number of studies pointed
out the contribution of HDACs in the modulation of lipid (Galmozzi et al., 2013,
Knutson et al., 2008, Sun et al., 2011) and BA metabolism and on the regulation
of CYP7A1 (Kemper et al., 2004, Ponugoti et al., 2007). In addition, previous
studies from our laboratory showed that HDAC inhibitors (HDAC-i) stimulate
CYP7A1 expression in vitro and in vivo by preventing the negative feedback
exerted by BA, consequently decreasing serum cholesterol in mice (Mitro et al.,
2007). Therefore, although the involvement of these enzymes in the regulation
of these processes has been demonstrated, several questions remain unsolved. It
is still unclear which class of HDACs is mainly involved, what is the contribution
of specific HDACs and corepressors in the regulation of CYP7A1 gene expression
and ultimately their impact on BA and cholesterol metabolism.
To elucidate this issues, first of all, in this study I examined the effects of
class-selective HDAC inhibitors on the promoter activity of hCYP7A1 in vitro. The
data obtained suggest a major involvement of class I HDACs in the negative
regulation of hCYP7A1, since class I selective HDAC inhibitor MS275 was able to
totally prevent the BA-induced repressive effect on hCYP7A1 promoter, while the
class II HDAC inhibitor MC1568 yielded only a partial, although significant,
recovery of gene transcription. The apparently lower contribution of class II
HDACs might be ascribed, on one hand, to the minimal histone deacetylase
activity of class IIa HDACs, the subclass involved in this regulation, due to a swap
Discussion
81
of a key tyrosine residue in the catalytic domain with a histidine (Lahm et al.
2004); in fact, they mainly act as scaffold molecules to recruit class I HDACs
(Nebbioso et al., 2009), thence the inhibition of their deacetylase activity does
not impair their actual function. On the other hand, MC1568 has been well
described as inhibitor of HDAC4 and HDAC5 (Nebbioso et al., 2009, Scognamiglio
et al., 2008), but the role on HDAC7, the most involved in CYP7A1 regulation
among class IIa HDACs, has not been clarified. Therefore, this might explain the
only partial recovery of gene expression that we observe after class II selective
MC1568 treatment.
Moreover, the RNAi approach in the human hepatoma reporter cell line,
HepG2 2.2.1 luc, allowed to investigate the contribution of HDAC1, HDAC3 and
HDAC7, identified by Mitro et al. as main constituents of the repressive complex
formed on hCYP7A1 promoter upon BA treatment (Mitro et al., 2007). Although
the silencing of these HDACs was able to significantly increase the basal level of
hCYP7A1, pointing out their relevance in the regulatory mechanism of this gene,
only HDAC1 and HDAC7 knock-down prevented the BA-induced inhibition of
hCYP7A1 transcription, highlighting their peculiar role in this pathway. The
stronger effect of HDAC1 deletion might be partially due to the loss of
interaction of HDAC1 with SHP that weaken SHP repression on CYP7A1 gene
transcription as well; in fact, it has been demonstrated that induction of SHP
plays its repressive effect by recruiting Swi/Snf-Brm chromatin remodeling
complex, which also contains an mSin3A/HDAC-1, to hCYP7A1 promoter (Kemper
et al., 2004).
In light of the possible differences between human and mouse regulation
of cholesterol catabolism, in this study I also pinpointed the role of specific
HDACs and corepressors firstly in vitro on murine primary hepatocytes and then
in vivo on C57BL/6J mice. In the in vitro study I knocked-down Hdac1, Hdac3,
Hdac7 and Smrt. I also knocked down Hdac4 and Hdac5, since it was previously
described their possible compensatory role in the absence of Hdac7 (Mihaylova et
al., 2011). Our results underscore the major contribution of Hdac1, Hdac7 and
Smrt in the repression of Cyp7a1 mRNA levels. These evidences suggest HDAC7,
Discussion
82
HDAC1 and SMRT as essential actors in the formation of the repressive complex
acting on Cyp7a1 promoter, indicating that their recruitment is critical to the
feedback inhibition of Cyp7a1 gene expression induced by BA.
The effects observed in vitro on murine primary hepatocytes were also
confirmed in vivo in C57BL/6J mice, where AdHdac7 and AdSmrt significantly
increase Cyp7a1 mRNA levels, thus confirming their important role in the
regulation of BA metabolism in vivo as well. However, AdHdac1 was ineffective in
silencing its target gene in vivo, therefore further studies will be necessary to
assess its contribution in the regulation of Cyp7a1 in vivo. In spite of the
elevated expression of Cyp7a1 gene upon administration of AdHdac7 and AdSmrt,
no changes of plasma cholesterol levels were observed. This apparent
discrepancy is actually ascribable to the adenoviral infection protocol. To avoid
the onset of inflammatory response in the liver a single dose of Ad was injected
to each mouse and after 5 days mice were sacrificed. Hence, only the acute
effect of Hdac7 and Smrt deletion on Cyp7a1 expression could be observed
whereas it was not possible to detect changes of plasma cholesterol levels, as
they would have need longer time and multiple injections.
Both the in vitro and the in vivo evidences obtained after silencing,
highlighted the role of the corepressor Smrt in the regulation of Cyp7a1 gene
expression. It has been well established, in fact, that NCoR and Smrt recruit
HDACs and they repress gene transcription by interacting with class I and class II
HDACs (Kao et al., 2000, Huang et al., 2000). It has been demonstrated that Smrt
contains a histone interaction domain (HID) and a deacetylase activation domain
(DAD) that binds and activates HDAC3, promoting the recruitment of repressive
complex on target gene (Yu et al., 2003). Furthermore, it has been demonstrated
that corepressors SMRT/N-CoR and mSin3A associate with Hdac7 in yeast and in
mammalian cells, suggesting association of multiple repression complexes (Kao
et al., 2000). In line with these evidences, this study suggests a peculiar role of
the corepressor Smrt that, together with Hdac7 translocating in the nucleus after
BA administration, might function as a platform in the formation of the
repressive complex on Cyp7a1 promoter.
Discussion
83
These evidences together with previous results published by our laboratory
showing that HDAC7 shuttles from cytoplasm to nucleus in response to BA and
represses CYP7A1 transcription (Mitro et al., 2007), suggest a key role of HDAC7
in cholesterol and BA metabolism. Therefore, to investigate more extensively the
contribution of Hdac7 in the regulation of Cyp7a1 and its implication in
cholesterol homeostasis in vivo, I used liver specific Hdac7 conditional knock-out
mice (H7LivKO). A previous study by Chang and coworkers demonstrated that
Hdac7 is expressed in the vascular endothelium during early embryogenesis and
played a crucial role in angiogenesis since it represses matrix metalloproteinase
(MMP) 10 expression that degrades the extracellular matrix and therefore it
maintains vascular integrity. In HDAC7 knock-out mice, the lack of this inhibition
causes loss of endothelial cells adhesion, vascular dilation and rupture resulting
in embryonic lethality by E11 (Chang et al., 2006). Therefore, to overcome the
embryonic lethality, I generated a liver specific knock-out mouse, lacking Hdac7
only in the liver. H7LivKO mice were fed Western diet, a diet enriched in
cholesterol, to increase the low basal level of LDL cholesterol of wt mice and to
emphasize the potential effect of Hdac7 loss on BA and cholesterol metabolism.
The potential role of the hepatic Hdac7 in the regulation of Cyp7a1 gene and the
impact of its ablation on BA and cholesterol homeostasis in vivo has not been
investigated previously. This study demonstrated that Hdac7 is deeply involved in
the molecular regulatory pathway of Cyp7a1 gene expression thus affecting
cholesterol metabolism and lipoprotein profile. Hdac7 ablation significantly
increased Cyp7a1 transcription leading to higher content of BA in the liver and in
plasma. Of note, fecal excretion of major BA, an index of increased BA
biosynthesis, was higher in H7LivKO mice on Western diet and is likely to explain
the reduction of liver and plasma cholesterol compared to control mice on
Western diet. The importance of bile acid homeostasis in the resistance of High
Fat Diet (HFD)-induced obesity and of hyperlipidemia in mice overexpressing
Cyp7a1 has been previously well described (Li et al., 2010b). In accordance to
the evidences showed by Li and coworkers, our study reveals that the induced
Cyp7a1 expression by the ablation of Hdac7 and the consequent increased BA
Discussion
84
biosynthesis result in improvement of cholesterol and lipid homeostasis and in
lipoprotein profile. Despite similar food consumption, H7LivKO mice showed 12%
reduction of body weight compared to control. This might be a consequence of
the higher circulating BA, which activate TGR5, the membrane BA receptor
localized in brown adipose tissue, and increase energy expenditure via
deiodinase 2 (D2) activation that causes increase of 3,5,3′-tri-iodothyronine (T3)
and consequently uncoupled protein 1 (UCP1) induction, a well known activator
of non-shivering thermogenesis (Watanabe et al., 2006); consistent with this
evidence the increased pool of BA observed in Cyp7a1-tg mice induce fatty acid
oxidation genes in brown adipose tissue (Li et al., 2010b). Furthermore, H7LivKO
mice were protected against fatty liver induced by Western diet. The
morphological evidence was supported by the significant decrease of hepatic
cholesterol of Western diet-H7LivKO mice, a main component of lipid droplets in
hepatic steatosis, even though the total hepatic triglycerides content displayed
only a mild but not significant reduction. It is possible that reduced hepatic
triglycerides content could be emphasized with a targeted analysis of single fatty
acids.
A further evidence of phenotipical improvement of liver in H7LivKO mice
pointed out by this study was the increased levels of hydrophilic BA in the liver,
such as α-MCA, β-MCA, ω-MCA and UDCA. Several publications underscore the
association between hydrophobic BA and liver damage; a more hydrophobic BA
pool was observed in TGR5 KO mice leading to liver injury (Péan et al., 2013); in
addition, mice fed with a lithocholic acid–enriched diet exhibit a hydrophobic
bile composition and bile duct obstruction leading to destructive cholangitis with
bile infarcts (Fickert et al., 2006).
Another important evidence highlighted by this study was the reduction of
LDL-cholesterol in H7LivKO mice fed Western diet. Interestingly, an important
change also occured in HDL cholesterol. FPLC and agarose electroforesis analysis
suggest the presence of smaller and less rich in cholesterol HDL poarticles that
might resemble pre-beta HDL in H7LivKO mice. Since HDLs are major cholesterol
Discussion
85
carriers in mice, this evidence probably has a major impact on cholesterol
profile.
In fact, nascent or pre-beta HDL are a subset of HDL particles poor in
lipids that are critical in collecting cholesterol from peripheral tissues as the first
step in reverse cholesterol transport (Barter and Rye, 1996). They function as
cholesterol acceptors from either cells or apoB-containing lipoproteins, by the
lecithin:cholesterol acyltransferase (LCAT)-mediated esterification of cholesterol
becoming mature, lipid-rich, and spherical HDL alpha (von Eckardstein et al.,
2001). The higher presence of pre-beta HDL and the lower alpha HDL content in
H7LivKO mice fed Western diet, thus might suggest a lower cholesterol
deposition in peripheral tissues due to higher capacity to remove cholesterol
from peripheral cells.
CONCLUSIONS
Conclusions
87
This study elucidated the importance of specific HDACs and corepressors
in the regulatory machinery underlying CYP7A1 expression, highlighting HDAC1,
HDAC7 and SMRT as important players in this pathway. Moreover, it suggests
HDAC7 as a crucial actor of this pathway, affecting cholesterol and lipid profile
in vivo. The results obtained with the investigations performed during my
doctorate training represent a step forward contributing to shed light on the
mechanisms of CYP7A1 regulation and their impact in lipid homeostasis. It
remains to be elucidated the mechanism underlying the phenotypical
improvement caused by the ablation of Hdac7. ChIP-seq experiments will help us
to verify if the absence of HDAC7 impairs the recruitment of HDACs and SMRT in
the transcriptional repressive complex on Cyp7a1 promoter and it might suggest
the involvement of other transcriptional coactivators or corepressors, in turn
regulated by Hdac7, which can contribute to the phenotypical evidences
observed in H7LivKO mice. Moreover, it will allow to investigate the possible
cross talk between HDAC7 and FGF15/19 pathway in light of previous evidences
that showed that HDAC inhibitors prevent the repressive effect of FGF15/19 on
Cyp7a1 promoter. Moreover, microarray analysis of H7LivKO mice compared to wt
will reveal the effects of liver-specific HDAC7 ablation on global hepatic gene
expression and it will elucidate possible changes in the expression of genes
involved in lipid and lipoprotein metabolism that might be affected by the
absence of HDAC7. In addition, H7LivKO serum will be analyzed by bidimensional
electrophoresis and by quantitative FPLC to deepen lipoprotein changes.
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